Multi-lineage hepatic organoids reveal toxic exosome mediated indirect hepatotoxicity

Due to the paucity of effective evaluation tools and limited mechanistic understanding, indirect hepatotoxicity remains a formidable challenge in the realm of drug development. Here, we constructed a 3D multi-lineage hepatic organoids (3D MLHOs) comprising five major types of liver cells derived from human embryonic stem cells (hESCs) and identified imipramine (IMP) as an inducer of indirect hepatotoxicity upon screening of 58 hepatotoxic drugs. Our research indicates IMP-induced hepatotoxicity is mediated by the specific interaction between IMP and the tyrosine kinase receptor B (TRKB) on non-parenchymal cells (i.e., hepatic stellate cells, HSCs), which in turn induces HSCs to release toxic exosomes (Toxic-EXOs) through the p53/hnRNPA2B1/DGCR8 pathway, ultimately leading to hepatocytes (HEPs) damage. The Toxic-EXOs are shown to cause hepatotoxicity by transferring microRNA-34a-3p (miR-34), a microRNA implicated in apoptotic pathways to HEPs. In HEPs that have taken up Toxic-EXOs, miR-34 disrupts cellular homeostasis by downregulating XIAP, an inhibitor of apoptosis protein, thereby activating caspase-3 and leading to cell apoptosis. Similarly, IMP long-term gavage induces HSCs apoptosis and indirectly causes hepatotoxicity in HEPs via toxic exosomes, without directly damaging hepatocytes. Our findings provide an invaluable biomimetic organoid platform for precision drug-testing and elucidate the indirect hepatotoxic mechanism, underscoring the importance of intercellular communication in drug-induced liver injury.